Inspecting the Triazole Scaffold as Powerful Antifibril Agents against 2N4R Tau and α-Synuclein Aggregates

Abstract

Alzheimer's (AD) and Parkinson's (PD) disease are neurodegenerative disorders that are considered to be a significant global health challenge due to their increasing prevalence and profound impact on both individuals and society. These disorders are characterized by the progressive loss of neuronal function, leading to cognitive and motor impairments. A key pathological feature of AD and PD is the abnormal accumulation of misfolded proteins within the brain. In AD, amyloid-beta aggregates into plaques, while tau proteins form neurofibrillary tangles (NFTs). Parkinson's disease, on the other hand, is marked by the accumulation of α-synuclein (α-syn) in the form of Lewy bodies (LBs). These protein aggregates are involved in neuronal dysfunction and neurodegeneration, contributing to disease progression. Research efforts are increasingly focused on identifying small molecules that can simultaneously target multiple pathological processes, offering the potential to not only alleviate symptoms but also modify the progression of neurodegeneration. Herein, a novel group of triazole-based compounds was designed and synthesized to curtail the aggregation of α-syn and tau proteins, which are closely linked to the physiopathology of PD and AD, respectively. A thioflavin T (ThT) fluorescence assay was used to measure fibril formation and assess the antiaggregation effects of various compounds. To further validate these findings, transmission electron microscopy (TEM) was employed as a direct method to visualize the impact of these compounds on fibril morphology. Inhibition of oligomer formation was evaluated using photoinduced cross-linking of unmodified proteins (PICUP), enabling the detection of early protein aggregation events. During fibril formation assays, three compounds (3e, 4b, 4d) demonstrated superior inhibitory activity as assessed by ThT fluorescence and TEM imaging. Subsequent evaluations, which included tests for antioligomer, anti-inclusion, and disaggregation effects identified compound 4d as the most promising candidate overall.

Figure 1

Design of the synthesized compounds as dual antifibrillar agents. (A) Antifibrillar agents I²⁴ and II²² containing indole scaffold previously published from Dr. Fortin laboratory; (B) Polyphenol catechin (−)-epi-gallocatechin-3-gallate (EGCG); and (C) designed compounds in this study.

Scheme 1. Synthetic Procedure for the Target Compounds 3a–h and 4b–d

Reagents and conditions: (a) NaNO₂, NaN₃, HCl:H₂O (1:1), 0-5°C, 3 h, 85-100% yield; (b) (i), PdCl₂(PPh₃)₂, CuI, TEA, DMF, r.t., 5–8 h; (ii) aq. NaOH, MeOH, r.t., 1 h, 72% yield (over the two steps); (c) Na ascorbate, CuSO₄.5H₂O, THF:H₂O (1:1), r.t., 71-91% yield; (d) BBr₃, DCM, 0°C, overnight, 8-12% yield.

Figure 2

Kinetics of α-syn formation with several triazole based-compound and dose–response of the lead, compound 4d. (A) Thioflavin T (ThT) kinetic curves illustrating the fibrillation process of α-syn (2 μM) with the thiazole compounds (100 μM). The control condition included only the vehicle (0.25% DMSO). For each experimental condition, triplicate measurements were taken at ten consecutive time points during the plateau phase of the fibrillation process. (B) Monomeric α-syn (6 μM) was subjected to variable concentrations of compound 4d to achieve the dose–response curve. Using Prism GraphPad, a nonlinear regression was applied in the semilog graph and the following equation was obtained Y = 103.3–51.33 *log(X) with sum of squares of 13.30. The trend line is indicated with a blue color. The dose–response curve exhibited a clear, dose-dependent linear relationship, indicating the compound’s potency in modulating the biological response across the tested concentration range of 6.25, 12.5, 25, 50, and 100 μM at 37 h incubation.

Figure 3

Lead 4d, a polyphenol triazole-based compound, reduced the aggregation of tau nonphosphorylated and phosphorylated 4R isoform by about 50%. The ThS fluorescence curves of 4d illustrate the aggregation kinetics of (A) tau 0N4R (6 μM), (B) tau 2N4R (12 μM), and (C) p-tau 1N4R (6 μM) in PBS-treated with chelex beads. The solution was supplemented with 150 μM heparin, 5 mM dithiothreitol (DTT), 40 μM ThS, and 10 mg/mL arachidonic acid in order to induce the aggregation. The control condition contained 0.25% DMSO, while compound 4d was tested at a concentration of 100 μM. The data represented by each curve are an average of three independent replicates.

Figure 4

Lead compound 4d exhibited a concentration-dependent inhibitory effect on α-syn oligomerization. (A) Compound 4d effectively prevented the formation of α-syn oligomers induced by tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate([Ru(bpy)₃]³⁺) and ammonium persulfate under brief light exposure (1 s) in the PICUP cross-linking assay. For this assay, α-syn (30 μM) was cross-linked in the presence of 100 μM of the lead compound. The samples prepared were not incubated for any substantial time prior PICUP experiments. In the control sample, which contained 0.25% DMSO without the test compound, higher molecular weight α-syn oligomers were clearly observed on Coomassie blue-stained polyacrylamide gels. In contrast, additional control samples that were either not exposed to light or lacked the cross-linking agent ([Ru(bpy)₃]³⁺) did not show any cross-linked products. (B) Monomeric α-syn (30 μM) was subjected to varying concentrations of the lead compound followed by PICUP cross-linking assay. Samples were loaded on a 16% polyacrylamide SDS-Page gel electrophoresis and stained with Coomassie blue to reveal the cross-linking products. The concentration-dependent α-syn antioligomer effect of lead compound 4d was confirmed. As the concentration of compound 4d decreased, there was a corresponding increase in the intensity of the high molecular weight bands (corresponding to oligomer). DMSO (0.25%) was used as a control, showing no significant impact on oligomer formation. The percentage of oligomer reduction is calculated by dividing the oligomer pixel density located between 35 and 40 kDa (most likely corresponding to dimer or mixture of higher order polymers) of the lane with compound treatment by the oligomer pixel density of the control lane (without the compound) and then multiplied by 100. The resulting value is then subtracted from 100 to determine the reduction.

Figure 5

Lead compound 4d exhibited a tau 0N4R antioligomer effect at high micromolar concentration. The tau 0N4R high molecular weight band corresponding to oligomer was observed above 180 kDa after loading samples subjected to the PICUP cross-linking assay. Tau (6 μM) in the presence of and ammonium persulfate was cross-linked under light exposure at a duration of 15 s. The samples were not incubated with the compounds prior the light exposure. The percentages of oligomer or monomer appearance were calculated by dividing the pixel density of the lane with compound treatment by the pixel density of the control lane (without the compound) and then multiplied by 100.

Figure 6

Compound 4d inhibits tau 0N4R oligomer formation and preserves monomeric species. (A) Percentage of residual tau 0N4R monomer remaining after treatment with compound 4d. The dose–response effect was evaluated using the PICUP cross-linking assay at different compound concentrations (50, 100, 200, and 300 μM). (B) Percentage of tau 0N4R oligomers detected after treatment with compound 4d at varying concentrations (50, 100, 200, and 300 μM). The results demonstrated a reduction in oligomer levels and increase monomer levels with increasing compound concentration. Average and SEM from three independent experiments were plotted in histograms. Original data are presented in Figures S45–S47. The statistical significance of differences between compound-treated samples and the control (DMSO, no compound treatment) was assessed using Dunnett’s post hoc test with p < 0.01 as the significant level.

Figure 7

Transmission electron microscopy (TEM) evaluation demonstrated that compound 4d significantly inhibited α-syn fibril formation. α-Syn (6 μM) was incubated with 0.25% DMSO as the control (CTRL) (A, C) and treated with compound 4d at concentration of 100 μM (B, D). After an incubation period of approximately 37 h, the samples were visualized using TEM. At low magnification (2500), dense mats of fibrils were observed with the control (DMSO) and small clumps were rarely encountered with the compound 4d. At high magnification (40k), many fibrils were observed with the control (DMSO) and the treatment with compound 4d resulted in rare proto-fibrils (bedded chain pattern). Scale bars in the images A and B represent 2 μm. Scale bars in the images C and D represent 200 nm.

Figure 8

Areas covered by α-syn fibrils and proto-fibrils from the experiment presented in Figure 7. The surface covered by fibrils were measured using TEM images at a magnification of 2500. Areas covered by α-syn fibrils and proto-fibrils were compared between DMSO and compound 4d. ImageJ software was used to set up the scale, adjust threshold, and measure area in μm². Different sample preparations deposited on copper grids were used for the analysis and one representative picture was selected from each grid. Three images obtained from independent grids for the control (DMSO) were quantified. Five images from three independent grids were analyzed for the treatment with compound 4d. **p < 0.001 by unpaired t test, two-tailed p value.

Figure 9

Lead compound 4d reduced the formation of tau 2N4R fibril formation as validated by transmission electron microscopy (TEM). TEM was employed to directly assess the effect of compound 4d on the monomeric tau isoform 2N4R (tested at 12 μM). Samples were treated with either DMSO (0.25%) as a control or 100 μM of compound 4d and incubated at 37 °C for 5 days. Scale bars correspond to 200 nm, with images captured at 40k magnification.

Figure 10

Compound 4d and EGCG disaggregate and prevent the formation of amyloid beta1–40 (Aβ_1–40) fibrils. (A) Thioflavin T fibril formation kinetics of 21 μM Aβ_1–40 was assessed in the presence of compound 4d and EGCG at 25, 50, and 100 μM concentrations. ThT was used at a final concentration of 40 μM. (B) Maximum fluorescence intensity in percentage was obtained at the end of the kinetics (approximately at 86 h) to evaluate the effects of compound 4d on Aβ_1–40 fibril formation in comparison with the control (0.25% DMSO). (C) Similarly the maximum fluorescence intensity in percentage were plotted for the EGCG treatment and the control (0.25% DMSO). For those two histograms, compound 4d and EGCG treatment were statistically significantly different compared to the control (0.25% DMSO) at p < 0.001 using the one-way ANOVA, Dunnet’s posthoc test.

Figure 11

Compound 4d exhibited a moderate disaggregation effect in comparison to EGCG, as confirmed by transmission electron microscopy (TEM). The disaggregation effect of compound 4d was compared to EGCG. EGCG resulted non fibrillar structures. The nontreated control presented dense plaques comprised of fibrils. Amyloid beta (0.593 ± 0.095 mg/mL) solutions obtained from the brain of an AD patient were incubated with DMSO (0.25%; referred to as “CTRL”); compound 4d (at 50 μM); or EGCG (at 50 μM) for 5 days at 37 °C before preparation of Formvar/carbon supported copper grids and direct visualization by TEM. Pictures were acquired at 40K. The scale bars correspond to 200 nm.

Figure 12

Formation of α-Syn inclusion was inhibited by 4d. M17D cells expressing the inclusion-prone αS-3K::YFP fusion protein (dox-inducible) were treated with 0.1% DMSO (vehicle; “0 μM”) as well as 1.25, 2.5, 5, 10, 20, and 40 μM of compounds 4b and 4d at t = 24 h after plating. αS-3K::YFP expression was induced with doxycycline at t = 48 h. (A) Incucyte-based analysis of punctate YFP signals relative to 0.1% DMSO was done at t = 96 h (N = 3 independent experiments, n = 6–18 individual wells total (0 μM, n = 18; 40, 20, and 10 μM, n = 6; 5, 2.5, and 1.25 μM n = 12). (B) Same as panel A, but confluence fold changes relative to DMSO vehicle (0 μM) were plotted. (C) Representative IncuCyte images of reporter cells treated with vehicle vs 40, 20, 10, and 5 μM compound 119 and 137 (t = 96 h), green channel. Arrows indicate αS-rich YFP-positive inclusions. Scale bar, 50 μm. All data are presented as fold-changes relative to DMSO control +/– standard deviation. One-way ANOVA, Dunnett’s posthoc test; ∗, p < 0.05; ∗∗∗∗, p < 0.0001; ns, nonsignificant.